Efthymiou Anastasia, Shaltouki Atossa, Steiner Joseph P, Jha Balendu, Heman-Ackah Sabrina M, Swistowski Andrzej, Zeng Xianmin, Rao Mahendra S, Malik Nasir
1National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA.
J Biomol Screen. 2014 Jan;19(1):32-43. doi: 10.1177/1087057113501869. Epub 2013 Sep 9.
Rapid and effective drug discovery for neurodegenerative disease is currently impeded by an inability to source primary neural cells for high-throughput and phenotypic screens. This limitation can be addressed through the use of pluripotent stem cells (PSCs), which can be derived from patient-specific samples and differentiated to neural cells for use in identifying novel compounds for the treatment of neurodegenerative diseases. We have developed an efficient protocol to culture pure populations of neurons, as confirmed by gene expression analysis, in the 96-well format necessary for screens. These differentiated neurons were subjected to viability assays to illustrate their potential in future high-throughput screens. We have also shown that organelles such as nuclei and mitochondria could be live-labeled and visualized through fluorescence, suggesting that we should be able to monitor subcellular phenotypic changes. Neurons derived from a green fluorescent protein-expressing reporter line of PSCs were live-imaged to assess markers of neuronal maturation such as neurite length and co-cultured with astrocytes to demonstrate further maturation. These studies confirm that PSC-derived neurons can be used effectively in viability and functional assays and pave the way for high-throughput screens on neurons derived from patients with neurodegenerative disorders.
目前，神经退行性疾病快速有效的药物研发受到阻碍，原因是无法获取用于高通量和表型筛选的原代神经细胞。通过使用多能干细胞（PSC）可以解决这一限制，多能干细胞可从患者特异性样本中获取，并分化为神经细胞，用于鉴定治疗神经退行性疾病的新型化合物。我们已经开发出一种高效方案，以培养纯神经元群体，经基因表达分析确认，该方案适用于筛选所需的96孔板形式。对这些分化的神经元进行活力测定，以说明它们在未来高通量筛选中的潜力。我们还表明，诸如细胞核和线粒体等细胞器可以通过荧光进行活细胞标记和可视化，这表明我们应该能够监测亚细胞表型变化。对源自表达绿色荧光蛋白的PSC报告系的神经元进行实时成像，以评估神经元成熟的标志物，如神经突长度，并与星形胶质细胞共培养以证明进一步成熟。这些研究证实，PSC衍生的神经元可有效地用于活力和功能测定，并为对神经退行性疾病患者来源的神经元进行高通量筛选铺平了道路。
